Compounds and methods for inhibiting axillary malodour

ABSTRACT

Enzymes mediating in the release of compounds characteristic of human malodour and in particular axillary malodour, and compounds that inhibit said enzymes having the general formula (I)

This invention is concerned with methods, compounds and compositionsuseful for the prevention or suppression of human malodour, inparticular human axillary malodour.

It is known that fresh sweat is odourless and that odour is only formedupon contact of sweat with skin bacteria (for example bacteria of thegenera of Staphylococcus and Corynebacteria) and it is believed thatodourless molecules present in sweat are degraded by bacteria colonisingthe axilla. It is generally accepted (Labows et. al., Cosmet. SciTechnol. Ser. (1999), 20:59–82) that highly unpleasant malodour isreleased from fresh sweat mainly by the Corynebacteria genus ofbacteria. The principal constituents thought to be responsible formalodour include volatile steroids, volatile sulphur compounds andshort-chain, branched fatty acids.

It has been suggested to treat malodour by eradicating the bacteriaresponsible for causing the odour. Indeed, commercially availablecosmetic deodorants often contain antibacterial compounds that generallyinhibit the growth of skin microflora. Antibacterial compounds currentlyused in deodorant products include, for example Triclosan(2,4,4′-trichloro-2′hydroxy-diphenyl-ether). However, a draw-back to theuse of antibacterials is the potential for disturbing the equilibrium ofthe skin's natural microflora.

It has also been suggested to include compounds in a deodorant thatwould specifically target and suppress the biochemical reactions thattransform odourless precursors present in sweat into volatile malodoroussteroids or sulphur compounds. Specifically, there have been severalpublications concerned with the inhibition of enzymes that are thoughtto be responsible for the release of volatile steroids or volatilesulphur products. In this regard see U.S. Pat. Nos. 5,487,886; 5,213,791and 5,595,728 which describe amino acid β-lyase inhibitors for use indeodorants. These agents are thought to block the release of sulphurvolatiles from cysteine derivatives. U.S. Pat. Nos. 5,676,937 and5,643,559 describe inhibitors of bacterial exoenzymes, namelysulphatases and glucuronidases. These compounds are supposed to reducethe release of volatile steroids from the corresponding sulphates orglucuronides. Patent application WO 00/01355 describes inhibition ofsteroid reductases. Finally, in German patent applications DE 19858811A1and DE 19855956A1 the use of esterase inhibitors as deodorant activeingredients is described.

However, fatty acids, in particular short chain, branched fatty acidsare known to play a role in axillary malodour, and are particularly foulsmelling. Whereas WO 00/01356 attributes axillary malodour to thecatabolism of long-chain fatty acids and teaches the use of certainperfumes to inhibit such catabolism, the art does not reflect anappreciation of the enzymatic process resulting in the release ofmalodorous fatty acids, in particular short chain, branched fatty acidsand therefore does not teach how malodour from these sources may beprevented or suppressed.

The applicant has now discovered the mechanism of the release of fattyacids in sweat and has found an enzyme thought to be responsible fortransforming odourless precursor compounds found in sweat, intomalodorous fatty acids. The applicant has also found specific inhibitorsof the enzyme and screening tools for identifying potential inhibitors,and also methods and compositions for preventing or suppressingmalodour. These and other aspects of the present invention will becomeapparent to those skilled in the art from the following description.

The invention provides in a first aspect an enzyme that mediates in abiochemical process whereby essentially odourless precursor compoundsfound in sweat are cleaved to release malodorous compounds, particularlymalodorous fatty acids, more particularly malodorous short chain,branched fatty acids.

The enzyme of the present invention was isolated from the bacteria ofthe genus Corynebacteria that can be found colonising the axilla, inparticular certain Corynebacteria sp., more particularly Corynebacteriastriatum Ax 20 which has been submitted on the 26, Apr. 2001 to theInternational Depository Authority DSMZ-Deutsche Sammlung VonMikrooganismen Und Zellkulturen GmbH, D-38124 Braunschweig. TheAccession Number provided by the International Depository Authority isDSM 14267.

The enzyme has not heretofore been available in isolated form. By“isolated” is meant that the enzyme is removed from its originalenvironment, i.e. from the environment in which it is naturallyoccurring. The present invention therefore provides the enzyme inisolated form, more particularly in isolated, purified form. By“purified form” is meant at least 80%, more particularly greater than90%, still more particularly 95%, most particularly 99% or greater withrespect to other protein and/or nucleic acid contaminants. The enzymemay be characterised by the amino acid sequence set forth in SEQ IDNO: 1. However, also included in the scope of the invention are proteinsor polypeptides, e.g. enzymes that comprise amino acid sequences thatare substantially similar to the amino acid sequence as set forth in SEQID NO: 1. In its broadest sense, the term “substantially similar” whenused in relation to an amino acid sequence, means a sequencecorresponding to a reference amino acid sequence, wherein saidcorresponding sequence encodes for a polypeptide or protein, e.g. anenzyme having substantially the same structure and same function as theenzyme encoded for by the reference amino acid sequence. The percentageidentity in sequences may be for example, at least 80%, particularly atleast 90% and most particularly at least about 95% of the amino acidresidues match over a defined length of the molecule and includesallelic variations. Sequence comparisons may be carried out using aSmith-Waterman sequence alignment algorithm which is known in the art.

Partial amino acid sequences of the enzyme set forth in SEQ ID NO: 2;NO: 3, and NO: 4 comprise additional aspects of the invention.

The amino acid sequence set forth in SEQ ID NO: 1 may be derived fromthe open reading frame contained in SEQ ID NO: 5. Accordingly, theinvention provides in another of its aspects an isolated nucleic acid,for example set forth in SEQ ID NO: 5, encoding for an enzyme having anamino acid sequence set forth in SEQ ID NO: 1.

All sequence data may be obtained according to techniques commonly knownin the art.

An enzyme of the present invention may have a molecular weight of 43 to48 kDa. In particular, it may have an apparent molecular mass onSDS-PAGE of 48 kDa and an effective molecular mass of 43365 Da asdetermined by nano-ESI MS (electron spray ionisation mass spectrometry)analysis and also derived from the amino acid sequence.

An enzyme of the present invention mediates in a biochemical processwhereby essentially odourless precursor compounds are cleaved to releasemalodorous compounds characteristically found in sweat. The precursorcompounds are substrates that may generally be described as derivativesof L-glutamine, in particular L-glutamine derivatives wherein theN_(α)atom of the L-glutamine residue is acylated with a residue of amalodorous compound, in particular a fatty acid residue, moreparticularly a short chain, branched fatty acid residue. One example ofsuch a precursor compound that was isolated from human sweat has thestructure:

Cleavage of this substrate at the N_(α) position releases the3-hydroxy-3-methyl-hexanoic acid, itself having a pungent odour, whichdehydrates to give 3-methyl-3-hexenoic acid which is another keymalodour volatile in human sweat. Proteins or polypeptides, e.g. enzymesthat act to cleave substrates of the type referred to hereinabove torelease malodorous acids are within the ambit of the present invention.

An enzyme according to the present invention may be particularly activein relation to certain substrates. For example, it can recogniseN_(α)-acylated-L-glutamine substrates. However, it is not able to cleavesimilar acylated derivatives of related amino acids such as L-glutamate,L-asparate or L-asparagine; nor does it recognise substrates wherein theN_(δ) or the COOH group of the L-glutamine moiety is substituted.Furthermore, it is stereospecific, for example it recognises derivativesof L-glutamine and not the analogues derived from D-glutamine. Havingregard to the substrate specificity, an enzyme of the present inventionmay be described as an aminoacylase, more particularly, anN_(α)-acyl-glutamine-aminoacylase. The acyl group at the N_(α) atom canvary widely, and the enzyme may cleave substrates for a wide variety ofdifferent smelling and non-smelling acids and other compounds. It mayalso, in addition to amide bonds, cleave carbamate bonds at the N_(α)position thereby mediating in the release of an alcohol, CO₂ andL-glutamine. It may also cleave acylated derivatives of L-glutaminewhere the N_(α) atom has been replaced by an oxygen atom, i.e.oxo-glutamine-derivatives.

Further, the enzyme requires as a cofactor a zinc ion. In this respectand in its ability to cleave amide-bonds, it may be considered to berelated to the group of enzymes known as zinc-metallopeptidases. Morespecifically, since it may cleave an amide-bond situated next to aterminal carboxyl group, it may also be considered to be related to thegroup of enzymes known as the zinc-carboxypeptidases.

Whereas an enzyme of the present invention is highly selective for theglutamine residue of a substrate, as mentioned above, applicant hassurprisingly found that a wide variety of glutamine derivatives are ableto fit into the enzyme. For example applicant found that disparatesubstrates such as Nα-(3-hydroxy-3-methyl-hexanoyl)-L-glutamine,Nα(3-methyl-2-hexenoyl)-L-glutamine, Nα-lauroyl-L-glutamine,Nα-(11-undecenoyl)-L-glutamine, Nα-tetradecanoyl-L-glutamine,Nα-decanoyl-L-glutamine, Nα-phenylacetyl-L-glutamine,Nα-Carbobenzyloxy-L-glutamine (=Z-glutamine),Nα-3,7-Dimethyl-6-octenyloxycarbonyl-L-glutamine,Nα-(3-hexenyl)oxycarbonyl-L-glutamine, Nα-Butyloxycarbonyl-L-glutamine,Nα-(4-tert-Butylcyclohexyloxycarbonyl)-L-glutamine,Nα-2-Phenylethyloxycarbonyl-L-glutamine,Nα-(3-Methyl-5-phenylpentanoxycarbonyl)-L-glutamine,Nα-(2-Adamantan-1-yl-ethoxycarbonyl)-L-glutamine,Nα-(2-Adamantan-1-yl-methoxycarbonyl)-L-glutamine,Nα-[2-(2,2,3-trimethyl-cyclopent-3-enyl)-ethoxycarbonyl)-L-glutamine,and Nα-(4-methoxy-phenylsulfanylcarbonyl)-L-glutamine are all able to becleaved by enzyme. These findings, together with knowledge as to thenature of metallopeptidases, suggest that an enzyme of the presentinvention has a high specificity for glutamine at its so-called“S₁′-site”, but will accept a wide variety of substituents at theNα-atom of the substrate provided those substituents are sufficientlybulky and hydrophobic to be received into the so-called “S₁ site” of theenzyme. The terms “S₁ site” and “S₁′ site” as used herein relate to thesites on metallopeptidase enzymes as will be apparent to a personskilled in the art.

An enzyme described hereinabove represents a particularly preferredembodiment of the present invention. However, other bacterial strains,for example other strains of Corynebacteria, or bacteria of the genusStaphylococci found in the microflora of the axilla also produce relatedenzymes that themselves mediate in biochemical reactions whereinL-glutamine derivatives are cleaved at N_(α). However, these relatedenzymes specifically cleave precursor compounds to release straightchain fatty acids, which acids play only a minor role in typical axillamalodour. These related enzymes, and inhibitors thereof, also formembodiments of the present invention.

A further aspect of the invention comprises a method of isolating anenzyme described above. Enzyme of the present invention occursintracellularly and can be released from the cells by mechanicaldisruption of the cell envelope. Thus, an enzyme may be isolated fromcellular extracts obtained from wild-type bacterial strains, especiallyfrom strains of Corynebacteria isolated from the human axilla, inparticular Corynebacterium striatum Ax 20.

Alternatively, an enzyme may be manufactured by recombinant means andthe invention provides in another of its aspects such methods,recombinant vectors and their use as reagents in said manufacture, andprocaryotic or eurocaryotic host cells transformed with said vectors.

Thus, an enzyme may be produced by growing host cells transformed by anexpression vector comprising foreign nucleic acid that encodes for theenzyme under conditions such that it is expressed, and thereafterrecovering it according to known techniques. In a particular embodimentof the invention a nucleic acid fragment that encodes for the SEQ ID NO:5 or a substantially similar nucleic acid sequence coding for an enzymewith an amino acid sequence which is substantially the same as sequenceSEQ ID NO 1, is introduced into an expression vector by operativelylinking the nucleic acid to the necessary expression control regionsrequired for gene expression. The vector is then introduced into anappropriate host cell, e.g. a bacterial host cell, more particularlyE.Coli. Numerous expression vectors are known and commerciallyavailable, and the selection of an appropriate expression vector andsuitable host cells which they can transform is a matter of choice forthe skilled person. Examples of expression vectors and host strains aredescribed in T. Maniatis et al. (Molecular Cloning, cold spring HarborLaboratory, 1982), other examples of vector-host strain combinations arethe vector pPROTet.E133 in strain DH5_(α)PRO which may be obtained fromClontech (Palo Alto, Calif., USA) or the vector pBADgIIIA in strain TOP10, which may be obtained from Invitrogen (Groningen, The Netherlands).

Recombinant production of an enzyme according to the invention is notlimited to the production in bacterial hosts. Any other means known tothose skilled in the art of producing an enzyme based on a definedgenetic sequence may be used. Such methods include, for example theexpression in genetically modified yeasts, in insect cells transformedwith a modified baculovirus and in eucaryotic cell lines or the in vitrotranscription and translation.

The enzyme produced according to methods described above may be purifiedaccording to known techniques. Thus, host-cells containing the enzymemay be extracted to release the enzyme, e.g. by mechanical disruption ofthe cells or by osmotic shock. Thereafter, crude enzyme may be separatedfrom host cell debris and host cell protein and nucleic acidcontaminants using well known techniques such as precipitation andchromatography. Any of the chromatography techniques known in the artfor purifying proteins may be employed. For example, ion-exchange,hydrophobic interaction, reverse phase, and size exclusionchromatography steps may be employed in any suitable sequence.Optionally, after each chromatography step the eluted enzyme may befurther purified by filtration and concentrated using, e.g.ultrafiltration techniques.

In another aspect of the invention there is provided a method ofscreening compounds as inhibitors of an enzyme as hereinabove described.In particular, in order to identify inhibitory compounds, the enzyme orcells or cell extracts containing the enzyme, obtained from any of theabove described sources, may be incubated along with an appropriatesubstrate that is cleavable by the enzyme, and with potential inhibitorycompounds. An appropriate substrate may be selected from any of theclass of precursor compounds referred to hereinabove, in particular anN_(α)-acylated L-glutamine or carbamate of glutamine. Particular usefulsubstrates are N_(α)-3-methyl-3-hydroxy-hexanoyl-glutamine,N_(α)-lauroyl-L-glutamine (commercially available from Fluka, Buchs,Switzerland) and N_(α)-carbobenzyloxy-L-glutamine (Z-glutamine;commercially available from Aldrich, Buchs, Switzerland). After acertain time of incubation, which may be determined according to routineexperimentation, analysis may be performed by measuring the releasedacid or alcohol, or by measuring the amount of free L-glutamine. Aparticularly useful approach for high-throughput screening of potentialinhibitors may be to measure the release of free L-glutamine byderivatising the free N_(α) group with an amine-group derivatisingagent, which upon reaction with the amine group forms a chromophore or afluorescent molecule. Particularly useful in this regard may be the useof fluorescamine (commercially available from Fluka, Buchs, Switzerland)to form a fluorescent molecule upon reaction with L-glutamine. Finally,the cleavage of the L-glutamine-substrate may be compared to controlreactions and the potential of the test compounds to inhibit thereaction may thereby be quantified.

Having regard to the nature of the enzyme and the screening method setforth above the skilled person will be able to derive compounds that areinhibitors of the enzyme in its mediation in the biochemical reactionresulting in the release of malodorous compounds, and these inhibitorsform yet another aspect of the invention.

Potential inhibitors may be selected, by way of non-limiting example,from dithiols, which molecules are capable of strongly co-ordinating toan active-site zinc atom located on the enzyme. One example of such acompound is dithiothreitol (2,3-dihydroxy-butane-1,4-dithiol). Otherzinc chelators may be useful inhibitors; such chelating agents mayinclude o-phenanthroline, EDTA, Na-pyrithione,amino-tri(methylene-phosphonic acid), ethylene-diimino-dibutyric acid(EDBA), Ethylenediamine-2-2′-diacetic acid, pyridine-2,6 dicarboxylicacid, Diethylenetriamine pentaacetate, Ethylenediamine disuccinic acid,and N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine. A furthergroup of inhibitors may be selected from Nα-actl-L-glutamines orcarbamates of L-glutamine which introduce some steric hindrance into themoiety substituted at the N_(α) atom. These inhibitors may compete withthe natural precursor compounds found in sweat for the active zinc siteon the enzyme, but display a reduced tendency, or no tendency, relativeto the natural precursor compound, to cleave at the N_(α) atom.

Compounds of formula (I)

have been found to be particularly interesting inhibitors of the enzymeand these compounds form a preferred embodiment of the presentinvention.

In formula (I), Y represents a direct bond to X, or a divalent chainthat may contain carbon, oxygen or nitrogen atoms, and may comprisefunctionality such as amide functionality —CONH— provided that the chainis not cleavable by the enzyme under condition of use. Preferably thisdivalent chain contains no more than 3, and preferably no more than 2atoms in the chain.

X represents a zinc-chelating group, e.g. a group bearing carboxylicacid functionality, or more particularly a methylene thiol group (II),or a phosphinyl group (III)

As regards the group R₁, given the broad range of substituents that canfit into the S₁ site of the enzyme, the nature of this group may varywidely provided it is sufficiently hydrophobic and/or bulky to fit intothis site. Preferably, it represents a linear, branched or cyclic carbonchain having about 1 to 14 carbon atoms, more particularly about 4 to 14carbon atoms. The aforementioned chain may contain one or moreheteroatoms such as O, N or S, and it may also contain unsaturation. Thechain may support one or more substituents, for example amide, ester,keto, ether, amine or hydroxyl halogen, or aryl or heteroarylsubstituents which aryl or heteroaryl groups may support substituentsselected from amide, ester, keto, ether, amine, halogen, alkyl orhydroxyl. The term “aryl” or “heteroaryl” as used herein is preferably amono-cyclic or polycyclic group containing from 6 to 14 carbon atoms,and as appropriate one or more heteroatoms such as O, N or S. By way ofexample, any of the substituents attached to the acyl carbonyl group ofthe substrates mentioned above would be suitable as a group R₁.

More preferred groups R₁ may be selected from a C₁₋₁₄ alkyl, morepreferably a C₄₋₁₄alkyl, e.g. n-butyl or sec-butyl, or an alkyl grouphere-mentioned substituted with a phenyl group, or a phenyl groupsubstituted with any of the substituents referred to above, e.g. abenzylic group or a phenylethyl group.

More preferred compounds of formula (I) are those compounds wherein

-   -   Y is selected from a direct bond to X, C₁₋₃ alkylene, e.g.        methylene, —CONH—, or —NH—, and    -   X is selected from methylene thiol (II) or phosphinyl (III).

Most preferred compounds of the present invention are those compounds offormula (I) wherein

-   -   Y represents an amide group —CONH— when X is methylene        thiol (II) or Y represents a methylene group when X is        phosphinyl (III).

Compounds of formula (I) contain chiral atoms and as such they can existas diastereomeric mixtures or they may exist as pure stereo-isomers.Most preferred compounds have an S-configuration on the Glutaminemoiety, and in the case of the methylene-thiol containing compounds alsoan S-configuration at the chiral centre in this group is preferred.

Examples of most preferred compounds are

wherein R1′ is phenyl (5a); iso-C₃H₇ (5b); or n-C₃H₇ (5c); or,

wherein R1′ is phenyl (8a); iso-C₃H₇ (8b); or n-C₃H₇ (8c).

The compounds of the present invention may be synthesised by couplingtogether the amino acid residue, e.g. the glutamine residue with theresidue R₁—X—Y— according to methods known in the art using readilyavailable starting materials or reagents.

The N-(sulfamylacyl)amino acids exemplified as (5a–c) above may beprepared in a manner set forth in Scheme I of FIG. 1. The t-butyl esterof glutamine may be coupled in a classical procedure of liquid-phasepeptide synthesis using EDCI+HOBT with various acetylsulfamyl alkanoicacids (2) leading to (3). After deprotection of the t-butyl ester groupand hydrolysis of the acetylsulfamyl group, one may obtain the compounds(5). The various acetylsulfamyl alkanoic acids (2) may be obtained formthe corresponding alpha amino acids. Brominative deamination leads tothe removal of the alpha-amino functionality replacing it with a bromineatom with retention of configuration. Subsequent removal of the bromineatom with the potassium salt of thioacetic acid will provide a compound(2) with inversion of configuration.

The skilled person will appreciate that other N-acylated glutaminecompounds of the present invention may be synthesised in an analogousmanner using appropriate reagents to provide the desired R₁—X—Y-residue.

The phosphinic-type compounds may be prepared by condensation of adesired alkyl halide and phosphinic acid ammonium salt to give acompound (6), followed by a 1,4 addition of (6) to theethyl-2-(N-trityl)carboxamidoethyl acrylate as set forth in Scheme 2 ofFIG. 2. Deprotection of the ester group and the amide can be achieved inmanner known per se to form the desired compound. The acrylate compoundmay be formed according to a method set forth in Scheme 3 of FIG. 3. Theskilled person will appreciate that other compounds of the presentinvention can be produce by 1,4 addition of a residue bearing R₁—X—Y—with the aforementioned acrylate.

Further details regarding the synthesis of compounds of the presentinvention are disclosed in the Examples hereinbelow.

Applicant has disclosed a wide range of compounds with inhibitoryproperties. However, having regard to the wide substrate specificity ofthe enzyme of the present invention responsible for the release of themalodorous compounds found in sweat, and the reliable methodology foridentifying inhibitors of the enzyme, the applicant is able to provide anovel method in the suppression of body odours which methods form anadditional aspect of the invention.

Accordingly, the invention provides in another of its aspects, a methodof suppressing axillary malodour comprising the step of providing acomposition for application to a person in need of treatment, saidcomposition containing an inhibitor compound and dermatologicallyacceptable vehicle therefor, said compound being selected from ascreening of compounds for activity in the inhibition of the enzyme.

Compounds, which inhibit an enzyme of the present invention reduce theactivity of the enzyme and may lead to a significant reduction of therelease of malodour acids from odourless fresh sweat. Compounds of thepresent invention display inhibition of the enzyme at concentrationsranging from 10⁻³ to 10⁻⁸ Molar. The activity of the compounds asinhibitors may be measured in terms of either their IC₅₀ values or theirKi values, both of which measures are well known to the person skilledin the art. As is well known, the IC₅₀ value provides the concentrationof an inhibitor needed to reduce enzyme velocity by half at a givensubstrate concentration. This value is dependent on the affinity of thesubstrate for the enzyme which is reflected in the value K_(m) of thesubstrate. In this way, the Ki value may be determined for a givensubstrate and a given substrate concentration by measuring IC₅₀ and thencalculating according to the following formula

$K_{i} = \frac{{IC}_{50}}{1 + \frac{\lbrack{Substrate}\rbrack}{K_{m}}}$Ki values for certain preferred inhibitors are set forth in Example 8below.

Compounds of the present invention may be added to any cosmetic andpersonal care products such as sticks, roll-ons, pump-sprays, aerosols,deodorant soaps, powders, solutions, gels, creams, sticks, balms andlotions to enhance the deodorising effect of these products.

Accordingly, the present invention relates to the use of inhibitorcompounds in compositions for the elimination or suppression ofmalodour. The invention also relates to compositions comprising an odoursuppressing quantity of an inhibitor of the enzyme and dermatologicallyacceptable vehicles which are generally well known in the art ofcosmetic and personal care products and require no further elaborationhere. Preferably, a compound of the present invention may be employed insaid products in amounts of about 0.01 to 0.5% by weight.

In an alternative method of malodour prevention or suppression, insteadof, or in addition to, employing inhibitors that act to prevent orsuppress the activity of the enzyme, one may employ agents that reducethe expression of the enzyme in bacteria containing a gene coding forsaid enzyme. Such agents may be screened either using wild-type strainsor genetically engineered strains of the bacteria expressing the enzyme.If wild-type strains are used, the level of enzyme expression may bedirectly measured under various environmental conditions and uponaddition of potential inhibitory compounds. Alternatively, geneticallyengineered Corynebacteria that are transformed with a vector containinga reporter gene may be used. These vectors may contain the reporter geneunder the control of the regulatory sequences for the enzyme expression,which regulatory sequence is contained in the SEQ ID NO: 6 and whichforms another aspect of the invention. For this purpose, the regulatorysequence, or any part thereof, may be cloned upstream of the reportergene into a broad host-range vector able to transform Corynebacteria.The reporter gene may thereby be put under the regulatory control of thegenetic sequence which controls the expression of the enzyme. The vectorobtained in this way may then transformed into a strain ofCorynebacterium. Particularly useful vectors for this purpose aredescribed by M. P. Schmitt (Infection and immunity, 1997, 65(11):4634–4641) and by N. Bardonnet and C. Blanco (FEMS Microbiol Lett.,1991, 68(1):97–102). Particularly useful marker genes are lacZ (codingfor β-galacturonidase, gfp (coding for the green fluorescent protein),luxABCD (coding for bacterial luciferase) and gusA (coding forglucuronidase). The genetically engineered strain may then be grown inthe presence of a compound to be tested and the expression of the markergene may be measured by conventional methods. Compounds that lead to areduction in expression (i.e. reduce the level of mRNA) may reducemalodour formation by reducing the level of enzyme in the axilla.

There now follows a series of examples that serve to illustrate theinvention.

EXAMPLE 1 Isolation of New Malodour Acid and Precursors thereof fromHuman Sweat

Fresh axilla secretions were sampled from human panellists by washingthe axilla with 10% ethanol. The samples were extracted with MTBE toremove interfering lipids. The hydrophilic phases obtained from thewashings from several individuals were then pooled. This material waspractically odourless, but upon hydrolysis of sub-samples with 1 M NaOH,it produced typical axilla malodour. To identify the malodour volatiles,hydrolysed sub-samples were extracted and concentrated by solid phaseextraction and then analysed by GC-sniff. Peaks that were rated ashaving a strong odour and closely related to axilla malodour wereanalysed by GC-MS. The samples contained one particular peak of an acidvery typical of axilla malodour. Based on the MS data the most probablestructure of this peak was 3-hydroxy-3-methyl-hexanoic acid. Thisassumption was verified by synthesising this latter compound andcomparing its spectra and retention times to the GC-MS data of the majormalodour peak in the GC-sniff analysis. This new malodour compound isstructurally related to the known sweat malodour acid3-methyl-2-hexenoic acid, and it is transformed into this lattercompound by dehydration upon prolonged incubation.

To identify the precursor for this acid, the pooled non-hydrolysedsample was separated on a Superdex gel filtration column (Pharmacia,Uppsala, Sweden) using NH₄CO₃/NaCl as the elution buffer. Individualfractions of this separation step were tested for the content of amalodour precursor by hydrolysis with 1 M NaOH. One fraction developedstrong malodour upon hydrolysis and this malodour could be attributed tothe release of 3-hydroxy-3-methyl-hexanoic acid by GC-MS analysis. Thisfraction was subjected to LC-MS analysis. It contained one major masspeak of 274 Da and an additional peak at 256 Da. The mass spectrum ofthe former peak suggested a compound where the3-hydroxy-3-methyl-hexanoic acid is linked to one molecule ofL-glutamine (i.e. N_(α)-3-hydroxy-3-methyl-hexanoyl-L-glutamine), whilethe second peak could, based on its mass, correspond to the dehydratedanalogue N_(α)-3-methyl-2-hexenoyl-L-glutamine.N_(α)-3-hydroxy-3-methyl-hexanoyl-L-glutamine was then synthesised andits MS spectrum and retention time in the LC-MS-analysis compared to andfound identical with the compound isolated from natural sweat.

EXAMPLE 2 Isolation of Axilla Bacteria having the Ability to Cleave theMalodour Precursor Compound

The axillary flora of 8 panellists was isolated with the detergent-scrubmethod: A 6 cm² area of the axilla was scrubbed with a phosphate bufferat pH 7 containing 1% Tween 80. The samples were spread-plated ontryptic soy agar amended with 5 g/L of Tween 80 and 1 g/L of lecithin.Single isolates obtained after 48 h incubation were subcultured andcharacterised. A total of 24 individual strains were identified based oncolony and cell morphology, gram-reaction, lipophilic growth, lipasereaction and API identification kits (bioMerieux, France; coryneformswith the API coryne kit and cocci with the ID Staph 32 kit). The strainswere grown overnight in a liquid medium (Mueller-Hinton amended with0.01% Tween 80), harvested by centrifugation and resuspended to a finalOD₆₀₀ of 1 in a semi-synthetic medium (Per litre: 3 g KH₂PO₄, 1.9 gK₂HPO₄, 0.2 g yeast extract, 0.2 g MgSO₄, 1.4 g NaCl, 1 g NH₄Cl, 10 mgMnCl₂, 1 mg Fe₃Cl₂, 1 mg CaCl₂). Aliquots of this stationary culturewere then amended with a final concentration of 500 ppm ofN_(α)-3-hydroxy-3-methyl-hexanoyl-L-glutamine (5% stock solutiondissolved in methanol). After 24 h incubation (with shaking at 300 rpm;36° C.) the samples were extracted and the amount of released3-hydroxy-3-methyl-hexanoic acid was determined by capillary GC. Table 1gives the results for a subset of the strains tested. From these resultsit appears that among the Corynebacteria isolated from the axilla some,but not all, are able to release 3-hydroxy-3-methyl-hexanoic acid formthe synthetic precursor. The Corynebacteria which are able to conductthis biochemical reaction may be found in the group of the lipophilicand in the group of the non-lipophilic Corynebacteria. Therefore, aspecific enzyme only present in some bacterial strains seems to beresponsible for this cleavage. Since it releases axilla malodour theputative enzyme was named AMRE, which stands for ‘axillary malodourreleasing enzyme’. Apparently the tested Staphylococci are not able tocatalyse this reaction, which is in agreement with the observation, thatonly subjects with an axilla flora dominated by Corynebacteria producethe most typical axilla malodour (Labows et. al., Cosmet. Sci Technol.Ser. 1999, 20:59–82). However, when Nα-lauroyl-L-glutamine was used assubstrate in the same experiment, it was found that also otherCorynebacteria and some Staphylococci can release lauric acid from thissubstrate. It therefore appears, that most axilla bacteria have arelated enzyme, but that many can only release straight fatty acidswhich make a minor contribution to typical axilla malodour.

TABLE 1 Cleavage of the natural malodour precursor by axilla bacteria.3-hydroxy- 3-methyl- hexanoic acid Isolate Species assignment Lipophilic(*) released (ppm) Ax1 Staphylococcus capitis − 0 Ax6 Staphylococcusepidermidis − 0 Ax9 Micrococcus luteus − 0 Ax3 Corynebacterium bovis + 0Ax7 Corynebacterium group G + 0 Ax15 Corynebacterium jeikeium + 37.4Ax19 Corynebacterium jeikeium + 105.1 Ax20 Corynebacterium striatum −262.7 (*) Corynebacteria isolated from the human axilla may be separatedinto two classes based on their requirement for a source of fatty acidsin the growth medium.

EXAMPLE 3 Purification and Analysis of the Enzyme from Strains thatCleave Malodour Precursor Compounds

Corynebacterium striatum A×20 was selected to isolate and purify theenzyme responsible for the cleavage of the precursorNα-3-hydroxy-3-methyl-hexanoyl-L-glutamine. The strain was grown during48 h in Mueller-Hinton broth amended with 0.01% Tween 80. A total volumeof 2 L of culture was harvested by centrifugation. The pellet was washedin Buffer A (50 mM NaCl; 50 mM NaH₂PO₄/K₂HPO₄ buffer at pH 7) and thisbuffer was used throughout the whole purification procedure. The cellswere disrupted mechanically by vortexing them with glass beads (425–600μm, Sigma, St-Louis, USA) during 30 min at maximal speed. The crude celllysate was then fractionated by precipitation with an increasingconcentration of (NH₄)₂SO₄. The precipitate obtained between 50% and 80%saturation of (NH₄)₂SO₄ contained the active enzyme. This enrichedsample was dissolved in Buffer A and then sequentially passed over fourchromatography columns: DEAE Sepharose CL-6B anion exchange resin(Pharmacia, Uppsala, Sweden; elution with a linear gradient from 0 to800 mM KCI); Phenyl-Sepharose hydrophobic interaction resin (Pharmacia;elution with a linear gradient from 1000 mM to 0 mM of (NH₄)₂SO₄; Mono Qstrong anion exchange column on the FPLC system (Pharmacia; elution witha gradient from 0 to 800 mM KCl) and finally Mono P weak anion exchangecolumn on the FPLC (elution with a gradient from 0–800 mM KCl in a 50 mMBis-Tris buffer instead of Buffer A). After each column separation theactive fractions (determined by fluorescent activity assay withNα-lauroyl-L-glutamine as substrate, see example 8) were pooled and thendesalted and concentrated by ultrafiltration (Amicon membrane YM10,Millipore, Bedford, US). The resulting active fractions after the lastcolumn separation contained one major protein band with an apparentmolecular weight of about 48 kDa as determined by SDS-PAGE. Itseffective molecular mass was determined by nano-ESI MS analysis andfound to be 43365±5 Da. This enzyme retained all its activity ifincubated with PMSF (Phenylmethylsulfonylfluoride, Roche Biochemicals,Mannheim, Germany) and Pefabloc SC(4-(2-aminoethyl)-benzenesulfonylfluoride, Roche Biochemicals), whichare typical inhibitors for serin- and cystein proteases. On the otherhand it was completely inhibited by 1 mM of EDTA and o-phenantrolin.This inhibition could be reversed by the addition of 1 mM ZnCl₂. Thisindicates that the enzyme belongs to the class of zinc-dependentmetallo-peptidases, requiring a Zn atom as cofactor. Finally, the enzymewas subjected to LC-ESI-MS/MS analysis after tryptic digestion and toanalysis of its N-terminal amino acid sequence. This led toidentification of its N-terminal amino acid sequence (SEQ ID NO: 2) andto the sequence of two internal peptides (SEQ ID NO: 3; SEQ ID NO: 4).

EXAMPLE 4 Substrate Specificity of the Enzyme

To understand in detail the structural requirements of substrates, theenzyme extracted from Corynebacterium striatum A×20 was incubated with awide variety of said compounds related to the originally isolatedN_(α)-3-methyl-3-hydroxy-hexanoyl-L-glutamine present in sweat. Eachcompound was used at a concentration of 500 ppm in Buffer A, andanalysis of released acid or alcohol was done by capillary GC after 24 hof incubation. First, different modifications at the N-terminus weretested. It was found, that the enzyme can cleave such simple substratesas N_(α)-lauroyl-L-glutamine and N_(α)-carbobenzyloxy-L-glutamine(=Z-glutamine). From the latter it releases benzyl-alcohol. OtherN-lauroyl-amino acids and Z-amino acids (all obtained from Fluka andAldrich, Buchs, Switzerland) were thus tested, but it was found thatamong the 20 amino acids occurring in proteins, the enzyme only cleavesL-glutamine derivatives, and, to much lesser extent, L-alaninederivatives. The results of some of the substrates tested are summarisedin Table 2. Furthermore the enzyme can cleave other carbamates ofL-glutamine, also derivatives where the alcohol is a fragrance alcohol(for example citronellol, see Table 2 compound 5), and it can thereforebe used to release pleasant smelling molecules from precursors. Indeed,it has broad specificity for substituents at N_(α) as reflected incompounds 1–5 (below) and as discussed above. Finally, it isstereospecific and cannot cleave derivatives of D-glutamine (Table 2,compound 19), it requires a free COOH group of the L-glutamine and doesnot cleave derivatives in which this group is linked to methanol orglycin (Table 2, compounds 20 and 21). It also cannot cleave aderivative in which the N_(δ) of glutamine is further derivatised (Table2, compound 22).

TABLE 2 Substrate specificity of the enzyme Cleavage by Substrateenzyme¹  1 Nα-(3-hydroxy-3-methyl-hexanoyl)-L-glutamine ++  2Nα-lauroyl-L-glutamine +++  3 Nα-decanoyl-L-glutamine +++  4Carbobenzyloxy-L-glutamine ++  5N_(α)-3,7-Dimethyl-6-octenyloxycarbonyl-L-glutamine +++  6N-Lauroyl-L-aspartate −  7 Nα-Lauroyl-L-lysine −  8Nα-Lauroyl-L-arginine −  9 N-lauroyl-L-alanine + 10Carbobenzyloxy-L-alanine + 11 Carbobenzyloxy-L-glutamate − 12Carbobenzyloxy-L-asparagine − 13 Carbobenzyloxy-L-aspartate − 14Carbobenzyloxy-L-serine − 15 Carbobenzyloxy-L-tyrosine − 16Carbobenzyloxy-L-glycine − 17 Carbobenzyloxy-L-histidine − 18Carbobenzyloxy-L-leucine − 19 Carbobenzyloxy-D-glutamine − 20Carbobenzyloxy-L-glutamine-O-Me − 21 Carbobenzyloxy-L-glutamine- Gly-OH− 22 4-benzylcarbamoyl-2-(S)-benzyloxyamino-butyric acid − ¹− indicatesno cleavage, + indicates cleavage < 10%, ++ cleavage 10–50% and +++cleavage over 50%.

EXAMPLE 5 Isolation of the Gene Coding for the Enzyme

Based on the partial amino acid sequence analysis (see example 3),degenerated primers were designed and used to amplify a 350 bp and a 650bp fragment of the corresponding gene between the N-terminus (SEQ ID NO2) and the two internal peptide sequences (SEQ ID NO 3 and 4).Chromosomal DNA of A×20 served as template. The primer with the sequenceSEQ ID NO 7 successfully annealed at the sequence coding for theN-terminus and the primers with the sequence SEQ ID NO 8 and SEQ ID NO 9annealed within the sequences coding for the internal peptides. StandardPCR conditions were used, and the annealing temperatures were optimisedon a gradient cycler (T-Gradient, Biometra, Göttingen, Germany). Theamplified DNA was cloned into the vector pGEM-T Easy (Promega, Madison,USA) and the nucleotide sequence determined on the ABI-Prism model 310(PE Biosystems, Rotkreuz, Switzerland) using standard methods. Based onthe obtained sequence, specific nested oligonucleotides were designed toclone the upstream (SEQ ID NO 10 and 11) and downstream region (SEQ IDNO 12 and SEQ ID NO 13). Chromosomal DNA of A×20 was digested with SmaIand PvuII and ligated to the GenomeWalker Adaptor (ClontechLaboratories, Palo Alto, USA). The upstream and downstream regions werethen amplified as described in the instructions to the UniversalGenomeWalker™ kit (Clontech Laboratories, Palo Alto, USA), cloned intothe vector pGEM T-easy and the nucleotide sequence determined. With thetwo enzyme digests two upstream (450 bp and 1200 bp) and two downstreamfragments (1200 bp and 3000 bp) were obtained. The full coding sequenceof the enzyme (SEQ ID NO 5) as well as upstream (SEQ ID NO 6) anddownstream regions were contained in the cloned region. The deducedamino acid sequence of the open reading frame corresponding to theenzyme (SEQ ID NO 1) was compared to public protein sequence databases(Swissprot and GeneBank, bacterial sequences) and it was found to alignvery well to known aminoacylases, some carboxypeptidases and variousputative peptidases identified in genome sequencing projects. A numberof these enzymes are summarised into the peptidase family m40, alsoknown as the ama/hipo/hyuc family of hydrolases.

EXAMPLE 6 Heterologous Expression of the Gene Coding for the Enzyme

The full-length sequence of the open reading frame coding for the enzymewas amplified with PCR from chromosomal DNA of A×20 using specificprimers (SEQ ID NO 14 and SEQ ID NO 15). The amplified DNA fragment wasthen digested with the restriction enzymes NcoI and Hind III It was thenligated into the vector pBADIIIA (Invitrogen, Groningen, TheNetherlands) pre-digested with the same enzymes. The resulting plasmidpBADgIIIAMRE was transformed into the host strain E. coli TOP10(Invitrogen). This strain was grown in LB broth until it reached anoptical density of about 0.5 at 600 nm. The culture was induced witharabinose (0.2% final concentration) incubated for 4 h, harvested bycentrifugation and disrupted by ultrasonication. Enzyme assays withNa-lauryl-Glutamine as substrate were performed in Buffer A with anincubation time of 1 h and a substrate concentration of 500 ppm. Table 3gives the activity of extracts obtained from wild-type cells and fromextracts of the induced and non-induced modified strains expressing theenzyme.

TABLE 3 Heterologous expression of the enzyme in E. coli release oflauric acid from Lauryl-Glutamine, 1 h incubation E. coli Top 10/ E.coli E. coli pBADgIIIAMRE not Top 10 Top 10 induced induced 4 h afterinduction below detection 23 ppm 329 ppm

EXAMPLE 7 Low Throughput Screening for Inhibitors of the Enzyme

Extracts of A×20 were prepared by mechanical disruption as described inExample 3. The extract (0.5 ml corresponding to 2 ml initial cellculture) was added to 3.5 ml of Buffer A and amended with 40 μl ofsubstrate (Nα-lauroyl-L-glutamine, 5% stock solution in methanol).Parallel samples were additionally amended with potential inhibitorycompounds to give a final concentration of 0.5 and 5 mM. The sampleswere incubated for 2 h and then extracted with MTBE and HCI and analysedfor released lauric acid using capillary GC. By comparing the samplescontaining potential inhibitory compounds with control samples withenzyme and substrate only, the inhibition (%) was calculated. Table 4gives the result for selected zinc chelating compounds. The same assaywas also made either with purified enzyme from the wild-type strain (seeexample 3) or with extracts from E.coli Top 10/pBADgIII AMRE inducedwith arabinose (see example 6). Furthermore, the same assay was madeusing stationary phase living cells of A×20 instead of an isolatedenzyme preparation. In this case successful uptake of the inhibitorsinto the cells and inhibition are measured simultaneously.

TABLE 4 Inhibition by zinc chelators of the isolated enzyme and of theenzymatic activity in intact cells % inhibition of % inhibition enzymeactivity of the isolated in living cells enzyme 5 mM 5 mM 0.5 mMo-phenantrolin 90.3%  100%  100% 2,2′-bipyridyl n.d.   65% n.d.Aminotri(methylene-phosphonic 55.7% 76.6% n.d. acid)Ethylen-diimino-dibutyric 53.7% 44.3% n.d. acidEthylendiamine-2-2′-diacetic 58.3%  100% n.d. acidPyridine-2,6dicarboxylic acid 64.3%  100% n.d. N,N,N′,N′-tetrakis-(2-85.2% 87.9% n.d. pyridylmethyl)-ethylendiamine Dithiothreitol n.d.  100%  98%

EXAMPLE 8 High Throughput Screening for Inhibitors of the Enzyme

Potential inhibitory compounds were dissolved in Buffer A and aliquotsof the solutions of different inhibitors (10 μl) were distributed toindividual wells of a white microtiter plate. Purified enzyme obtainedfrom the strain E.coli Top 10/pBADgIII AMRE was diluted in Buffer A (200pg/ml final concentration) and added to the inhibitory compounds. After10 min preincubation, the substrate (Nα-lauroyl-L-glutamine) was addedto the individual wells to a final concentration of 0.05 mM. After 15min of incubation, the amino-group of the released L-glutamine wasderivatised by adding to each well of the microtiter plate 50 μl of afluorescamine stock solution (2.5 mM in acetonitrile; fluorescamineobtained from Fluka, Buchs, Switzerland). After 5 min the fluorescencein the wells of the microtiter plates was measured with an excitationwavelength of 360 nm and an emission wavelength of 460 nm. Thefluorescence of control wells with enzyme, substrate and DMSO only wasthen compared to the fluorescence in wells containing potentialinhibitors. By varying the inhibitor concentration, the IC50 value foreach compound was determined, and the Ki values were calculated.

TABLE 5 Ki values for compounds of formula (I) Compound Ki value (nM) 5a54 ± 1  5b 130 ± 10  5c 410 ± 20  8a 50 ± 3  8b 58 ± 4  8c 110 ± 10 

SYNTHESIS EXAMPLE 1

The following description is made with reference to Scheme 1 in FIG. 1

Synthesis of the Thiol Inhibitors Step 1—Synthesis of (2R)-2-Bromo-alkylcarboxylic acids (1)

In a synthesis based on Fisher, S. R. W.; Justus Liebigs Ann. Chem.,1957, 357 , (0.165 mol) of the corresponding D-α-aminoacid aresolubilised in 165 mL HBr 48% and 150 mL water. The reaction mixture iscooled to 0° C. and a solution of NaNO₂ (18.3 g , 1.6 eq) in 60 mL wateris added dropwise. The mixture is stirred for 2.5 h at room temperature,then concentrated to remove the acid vapour, extracted with Et₂O fourtimes. The organic layers are washed with water, NaCl sat., dried overNa₂SO₄, and concentrated under reduced pressure yielded compound 1 asoil used without further purification.

1a, R: PhCH₂: Yield 100% Rf: 0.43 (CH₂Cl₂/MeOH/AA 9/1/0.2) ¹H NMR (CDCl₃270 MHz): 7.3(m, 5H), 4.3(t, 1H), 3.1–3.5 (m,2H).

1b, R: iBu: Yield 92.6% Rf: 0.50 (CH₂Cl₂/MeOH/AA 9/1/0.5) ¹H NMR (CDCl₃270 MHz): 4.35(t, 1H), 2.0(t,2H), 1.8(m, 1H), 1.0(m, 6H)

1c, R: nBu: Yield: 100% Rf: 0.48 (CH₂Cl₂/MeOH/AA 9/1/0.5) ¹H NMR (CDCl₃270 MHz): 4.3(t, 1H), 2.1–1.8 (m,2H), 1.5 (m, 4H), 0.9 (t,3H).

Step 2—Synthesis of (2S)-2-Acetylsulfanyl alkyl carboxylic acids (2)

Compound 1 (0.165 mol) solubilised in 165 mL NaOH 1N (1 eq) is cooled at0° C. Potassium thioacetate (22.65 g, 0.198 mol, 1.2 eq) in 60 mL H₂O isadded dropwise and the reaction mixture is stirred for 16 h at roomtemperature. The preparation is acidified by addition of HCl 1N (pH 1–2)then extracted with AcOEt three times. The organic layers are washedwith water, NaCl sat., dried over Na₂SO₄, and concentrated under reducedpressure yielded compound 2 as orange oil used without furtherpurification.

2a, R: PhCH₂: Yield 96.0% Rf: 0.43 (Cyclohexane/AcOEt/AA 5/5/0.1) HPLCKromasil C18 5μ100A, 250×4.6 mm, CH₃CN—H₂O (0.05% TFA) 40-60 R_(t)=8.9min ¹H NMR (CDCl₃ 270 MHz): 7.3(m, 5H), 4.3(t, 1H), 3.1–3.5 (m,2H),2.2(s, 3H).

2b, R: iBu: Yield 91.0% ¹ H NMR (CDCl₃ 270 MHz): 4.2(t, 1H), 2.4(s,3H),1.9–1.5(m, 4H), 0.9(m, 6H)

2c, R: nBu: Yield: 94.5% ¹H NMR (CDCl₃ 270 MHz): 4.1(t, 1H), 2.3(s, 3H),1.9(m,1H), 1.65(m,1H), 1.3(m, 4H), 0.9 (t,3H).

Step 3—Synthesis of N-[(2S)-2-acetylsulfanyl alkanoyl]-(S)-glutaminetert-butyl ester (3)

Compound 2 (2.607 mmol), (S)-Glutamine tert-butyl ester hydrochloride(1.2 eq, 746 mg), EDCI (1.2 eq, 929 mg), HOBt (1.2 eq, 479 mg), Et₃N(1.2 eq, 438 μL) are stirred overnight in 10 mL THF/CHCl₃. The reactionmixture is concentrated under reduced pressure and diluted in H₂O/AcOEt.The organic layer is washed with NaHCO₃ sat. (2×), citric acid 10% (2×),NaCl sat., dried over Na₂SO₄, and concentrated.

The crude product is purified by HPLC Kromasil C18 5μ100A, 250×20 mm(CH₃CN/H₂O 0.05% TFA 40–60) yielded compound 3 as a solid.

3a, R: PhCH₂: Yield 32.4%, wt: 343 mg. HPLC Kromasil C18 5μ100A, 250×4.6mm, CH₃CN—H₂O (0.05% TFA) 50-50 R_(t)=7.88 min ¹H NMR (CDCl₃ 270 MHz):7.3–7.2(m, 5H), 4.4(m, 1H), 4.3(t, 1H), 3.2 (dd,1H), 2.8 (dd,1H), 2.2(s,3H), 2.1(m,2H), 1.8(m,2H), 1.4(s, 9H).

3b, R: iBu: Yield 74.3%, wt: 352 mg. HPLC Kromasil C18 5μ100A, 250×4.6mm, CH₃CN—H₂O (0.05% TFA) 50-50 R_(t)=6.48 min ¹H NMR (CDCl₃ 270 MHz):6.8(d, 1H), 4.4(m, 1H), 4.15(t, 1H), 2.4(s,3H), 2.3–1.5(m, 7H), 1.4(s,9H), 0.9(m, 6H)

3c, R: nBu: Yield: 80.7%, wt: 307 mg. HPLC Kromasil C18 5μ100A, 250×4.6mm, CH₃CN—H₂O (0.05% TFA) 60-40 R_(t)=6.75 min ¹H NMR (CDCl₃ 270 MHz):6.8(d, 1H), 4.4(m, 1H), 4.1(t, 1H), 2.4(s, 3H), 2.2–1.5(m, 10H), 1.4(s,9H), 0.9 (t,3H).

Step 4—Synthesis of N-[(2S)-2-acetylsulfanyl alkanoyl]-(S)-glutamine (4)

Compound 3 (0.58 mmol) is solubilized in 3 mL CH₂Cl₂ and 3 mL TFA areadded at 0° C. The reaction mixture is stirred for 3 h at roomtemperature. The solvent and excess reagent are eliminated under reducedpressure. The crude product is coevaporated 2 times with cyclohexaneyielded compound 4 as oil used without further purification.

4a, R: PhCH₂: Yield 100%, wt: 206 mg. HPLC Kromasil C18 5μ100A, 250×4.6mm, CH₃CN—H₂O (0.05% TFA) 30-70 R_(t)=8.67 min ¹H NMR (DMSO+TFA 270MHz): 8.5 (d, 1H), 7.2(m, 5H), 4.4(t, 1H), 4.05(m, 1H), 3.2 (dd,1H), 2.8(dd,1H), 2.2(s, 3H), 2.1(t,2H), 1.9(m,1H), 1.8(m, 1H).

4b, R: iBu: Yield 100%, wt: 299 mg. HPLC Kromasil C18 5μ100A, 250×4.6mm, CH₃CN—H₂O (0.05% TFA) 30-70 R_(t)=6.36 min ¹H NMR (DMSO+TFA 270MHz): 8.5(d, 1H), 4.3–4.0(m, 2H), 2.4(s,3H), 2.1(m, 2H)1.9(m, 1H),1.7(m, 2H), 1.5(m, 1H), 1.3(m, 1H), 0.9(d, 3H), 0.8(d,3H)

4c, R: nBu: Yield: 100%, wt: 261 mg. HPLC Kromasil C18 5μ100A, 250×4.6mm, CH₃CN—H₂O (0.05% TFA) 30-70 R_(t)=6.75 min ¹H NMR (DMSO+TFA 270MHz): 8.5(d, 1H), 4.1(m, 2H), 2.4(s, 3H), 2.1(t,2H), 1.9(m, 1H), 1.7(m,1H), 1.6(m, 1H), 1.2(m, 3H), 0.9 (t,3H).

Step 5—Synthesis of N-[(2S)-2-mercapto alkanoyl]-(S)-glutamine (5)

Compound 4 (0.38 mmol) is solubilized under argon in 2 mL degassed MeOHand 1.16 mL degassed NaOH (3 eq) are added. The reaction mixture isstirred for 2 h at room temperature. HCl 1N is added to obtain pH=1 andthe solvent is eliminated under reduced pressure. The product isextracted with AcOEt. After evaporation the product is solubilized inwater and lyophilised to give 5 as a white hygroscopic solid.

5a, R: PhCH₂: Yield 76.5%, wt: 91 mg. HPLC Kromasil C18 5μ100A, 250×4.6mm, CH₃CN—H₂O (0.05% TFA) 30-70 R_(t)=6.65 min SM-ES(+): [M+Na]⁺=333 ¹HNMR (DMSO+TFA 270 MHz): 8.5 (d, 1H), 7.2(m, 5H), 4.1(m, 1H), 3.6(q, 1H),3.1 (dd,1H), 2.7 (dd,1H), 2.1(t, 2H), 1.9(m,1H), 1.8(m, 1H).

5b, R: iBu: Yield 51.3%, wt: 133 mg. SM-ES(−): [M−H]⁻=275 HPLC KromasilC18 5μ100A, 250×4.6 mm, CH₃CN—H₂O (0.05% TFA) 30-70 R_(t)=5.36 min ¹HNMR (DMSO+TFA 270 MHz): 8.3(d, 1H), 4.1(m, 1H), 3.4(m, 1H), 2.1(m, 2H),2.0–1.3(m, 5H), 0.9(d, 3H), 0.8(d,3H)

5c, R: nBu: Yield: 74.3%, 168 mg. HPLC Kromasil C18 5μ100A, 250×4.6 mm,CH₃CN—H₂O (0.05% TFA) 30-70 R_(t)=5.89 min SM-ES(+): [M+Na]⁺=299 ¹H NMR(DMSO+TFA 270 MHz): 8.2(d, 1H), 4.2(m, 1H), 3.2 (q, 1H), 2.1(t,2H),1.9(m, 1H), 1.7(m, 1H), 1.6(m, 1H), 1.2(m, 3H), 0.8 (t,3H).

SYNTHESIS EXAMPLE 2

This synthesis is described with reference to Scheme 2 of FIG. 2:

Synthesis of the Phosphinic Inhibitors Step 1—Synthesis of Alkylphosphinic acids (6)

The synthesis is based on the method of Boyd, E. A.; Regan, A. C.;Tetrahedron Letters, 1994, 24, 4223. In a 100 mL flask equipped with aseptum and a condenser, 4.2 g (51.85 mol) ammonium phosphinate and HMDS(8.57 g, 53.08 mmol, 1.02 eq) are heated under N₂ at 100–110° C. for 2h. The reaction mixture is cooled at 0° C. and 50 mL dried CH₂Cl₂ isadded followed by the addition of the bromide derivative (53.08 mmol,1.02 eq). The mixture is stirred overnight at room temperature.

The precipitate is filtered and the filtrate concentrated under reducedpressure. The crude product is dissolved in CH₂Cl₂/MeOH. The precipitateis removed and the crude product is eluted on silica gel (CH₂Cl₂/MeOH/AA9/1/0.4) yielding compound 6.

6a, R: PhCH₂: Yield 43.3%, wt: 3.50 g. Rf: 0.21 (CH₂Cl₂/MeOH/AA 9/1/0.4)¹H NMR (DMSO+TFA 270 MHz): 7.2(m, 5H), 6.9(d, 1H), 3.1(dd, 2H)

6b, R: iBu: Yield 32.1%, wt: 2.86 g. ¹H NMR (DMSO+TFA 270 MHz): 6.9(d,1H), 3.6(m, 2H), 1.5(m, 1H), 0.9(m, 6H)

6c, R: nBu: Yield: 56.9%, wt: 3.60 g. ¹H NMR (DMSO+TFA 270 MHz): 6.9(d,1H), 1.6(m, 2H), 1.3(m, 4H), 0.8(t, 3H)

Step 2—Synthesis of2-(Benzyl-hydroxy-phosphinoylmethyl)-4-(trityl-carbamoyl)-butyric ethylester (7a)

This synthesis is based on the method of Boyd, E. A.; Regan, A. C.;Tetrahedron Letters, 1994, 24, 4223. In a 25 mL flask equipped with aseptum and a condenser, compound 6a (156 mg, 1 mmol) and HMDS (218 μL,1.02 eq) are warmed up under N₂ at 100–110° C. for 2 h. The reactionmixture is cooled at 0° C. and compound 14 (426 mg, 1.03 mmol) in 5 mLdried CH₂Cl₂ is added. The mixture is heated overnight at 60° C.

The reaction mixture is concentrated under reduced pressure. The crudeproduct is purified by HPLC Kromasil C18 5μ100A, 250×20 mm (CH₃CN/H₂O0.05% TFA 60-40) yielded 234 mg compound 7a as a white solid. Yield41.1%.

HPLC Kromasil C18 5μ100A, 250×4.6 mm, CH₃CN—H₂O (0.05% TFA) 70-30R_(t)=5.99 min ¹H NMR (DMSO+TFA 270 MHz): 8.5 (s, 1H), 7.2(m, 20H),4.1(q, 2H), 3.0(d, 2H), 2.6(m, 1H), 2.3(t, 2H), 2.0–1.6 (m, 4H), 1.1(t,3H)

Synthesis of2-(Alkyl-hydroxy-phosphinoylmethyl)-4-(trityl-carbamoyl)-butyric ethylester (7b–c)

Compound 6b or 6c (3.27 mmol) is solubilized in 3 mL CH₃CN. Compound 14(1.35 g, 3.27 mmol) and BSA (4.06 mL, 5 eq) are added and the reactionmixture is stirred 72 h at room temperature under N₂. The reactionmixture is concentrated under reduced pressure. The crude product ispurified by HPLC Kromasil C18 5μ100A, 250×20 mm (CH₃CN/H₂O 0.05% TFA60-40).

7b, R: iBu: Yield 5.3%, wt: 92 mg, white solid. HPLC Kromasil C185μ100A, 250×4.6 mm, CH₃CN—H₂O (0.05% TFA) 70-30 R_(t)=5.57 min ¹H NMR(DMSO+TFA 270 MHz): 8.6 (s, 1H), 7.2(m, 15H), 4.0(q, 2H), 2.6(m, 1H),2.2(t, 2H), 1.9 (m, 2H), 1.7(m, 3H), 1.5 (m, 2H), 1.1(t, 3H), 0.9(d, 6H)

7c, R: nBu: Yield: 8.9%, wt: 155 mg, white solid. HPLC Kromasil C185μ100A, 250×4.6 mm, CH₃CN—H₂O (0.05% TFA) 60-40 R_(t)=10.29 min ¹H NMR(DMSO+TFA 270 MHz): 7.2(m, 15H), 6.8(s, 1H), 4.1(q, 2H), 2.7(m, 1H),2.4–1.4(m, 12H), 1.2(t, 3H), 0.9(d, 3H)

Step 3—Synthesis of2-(Alkyl-hydroxy-phosphinoylmethyl)-4-(trityl-carbamoyl)-butyric acid(8)

Compound 7 (0.41 mmol) is solubilized in 2 mL EtOH and 2 mL LiOH 1N(5eq) are added. The reaction mixture is stirred for 2 h at roomtemperature. HCl 1N is added to obtain pH=1 and EtOH is removed underreduced pressure. The product is extracted by AcOEt. The organic layersare washed with NaCl sat., dried over Na₂SO₄ yielding compound 8 asoils.

8a, R: PhCH₂: Yield 95.0%, wt: 211 mg. HPLC Kromasil C18 5μ100A, 250×4.6mm, CH₃CN—H₂O (0.05% TFA) 40-60 R_(t)=7.70 min ¹H NMR (DMSO+TFA 270MHz): 8.5 (s, 1H), 7.2(m, 20H), 3.0(dd, 2H), 2.5(m, 1H), 2.3–1.6 (m, 6H)

8b, R: iBu: Yield 97.6%, wt: 81 mg. HPLC Kromasil C18 5μ100A, 250×4.6mm, CH₃CN—H₂O (0.05% TFA) 60-40 R_(t)=5.46 min ¹H NMR (DMSO+TFA 270MHz): 8.6 (s, 1H), 7.2(m, 15H), 2.6(m, 1H), 2.2(m, 2H), 1.9 (m, 2H),1.7(m, 3H), 1.5(m, 2H), 0.9(d, 6H)

8c, R: nBu: Yield: 69.2%, wt: 101 mg. HPLC Kromasil C18 5μ100A, 250×4.6mm, CH₃CN—H₂O (0.05% TFA) 70-30 R_(t)=3.92 min ¹H NMR (DMSO+TFA 270MHz): 8.6(s, 1H), 7.2(m, 15H), 2.7(m, 1H), 2.0–1.0(m, 12H), 0.8(d, 3H)

Step 4—Synthesis of2-(Alkyl-hydroxy-phosphinoylmethyl)-4-carbamoyl-butyric acid (9)

Compound 8 (0.197 mmol) is solubilized in 4 mL TFA in presence of 90 μLiPr₃SiH. The reaction mixture is stirred 2 h at room temperature. ExcessTFA is removed under reduced pressure and the reaction mixture isco-evaporated with cyclohexane (2×). The crude product is purified byHPLC Kromasil C18 5μ100A, 250×20 mm (CH₃CN/H₂O 0.05% TFA 30-70) yieldingcompound 9.

9a, R: PhCH₂: Yield 88.1%, wt: 52 mg, oily product. SM-ES(+): [M+H]⁺=300HPLC Kromasil C18 5μ100A, 250×4.6 mm, CH₃CN—H₂O (0.05% TFA) 50-50R_(t)=2.34 min ¹H NMR (DMSO+TFA 270 MHz): 7.2(m, 5H), 3.0(d, 2H), 2.5(m,1H), 2.0–1.6 (m, 6H)

9b, R: iBu: Yield 71.5%, wt: 30 mg, oily product. SM-ES(−): [M−H]⁻=264HPLC Kromasil C18 5μ100A, 250×4.6 mm, CH₃CN—H₂O (0.05% TFA) 60-40R_(t)=2.36 min ¹H NMR (DMSO+TFA 270 MHz): 2.6(m, 1H), 2.0–1.5(m, 9H),0.9(d, 6H)

9c, R: nBu: Yield: 98.0%, wt: 51 mg, oily product. SM-ES(−): [M−H]⁻=264HPLC Kromasil C18 5μ100A, 250×4.6 mm, CH₃CN—H₂O (0.05% TFA) 70-30R_(t)=3.92 min ¹H NMR (DMSO+TFA 270 MHz): 2.5(m, 1H), 2.1–1.1(m, 12H),0.8(d, 3H).

SYNTHESIS EXAMPLE 3

This synthesis is described with reference to Scheme 3 of FIG. 3.

Synthesis of the Ethyl 2 [2-(N-trityl)carboxamido ethyl)]acrylate Step1—Synthesis of Diethyl 2-(2-tert-butyloxycarbonyl ethyl)malonate (10)

In a method based on Prabhu, K. R.; Pillarsetty, N.; Gali, H.; Katti, K.V.; J. Am. Chem. Soc., 2000, 122, 1554, a mixture of diethylmalonate(11.12 g, 10.55 mL, 69.46 mmol), tert-Butylacrylate (10.17 mL, 69.46mmol, 1 eq), K₂CO₃ (9.60 g, 1 eq), nBu₄NHSO₄ (258 mg 0.01 eq) in 40 mLtoluene are heated under reflux during 16 h. The reaction mixture isfiltered, concentrated under vacuum yielded 19.6 g compound 10 as oilused without further purification.

Yield: 98.0% HPLC Kromasil C18 5μ100A, 250×4.6 mm, CH₃CN—H₂O (0.05% TFA)70-30 R_(t)=8.72 min ¹H NMR (CDCl₃ 270 MHz): 4.1 (q, 4H), 3.3 (t, 1H),2.2 (m, 2H), 2.05 (m, 2H), 1.3 (s, 9H), 1.1 (m, 6H).

Step 2—Synthesis of Diethyl 2-(2-carboxyethyl)malonate (11)

To a solution of compound 10 (19.6 g, 68.05 mmol), in 400 mL CH₂Cl₂ isadded 400 mL of TFA. The mixture is stirred under during 48 h at roomtemperature. The reaction mixture is concentrated under vacuum,coevaporated two times with cyclohexane to eliminate excess TFA yielded15.8 g compound 11 as oil used without further purification.

Yield: 100.0% ¹H NMR (CDCl₃ 270 MHz): 8.7(s, 1H), 4.2 (q, 4H), 3.4 (t,1H), 2.5 (m, 2H), 2.1 (m 2H), 1.2 (m,6H).

Step 3—Synthesis of Diethyl 2-(2-N-tritylcarboxamidoethyl)malonate (12)

In a method based on Haynes, R. K.; Starling, S. M.; Vonwiller, S. C.;J. Org. Chem., 1995, 60, 4690, compound 11 (15.79 g, 68.10 mmol) in 12mL thionyl chloride is heated under reflux during 1 h. The reactionmixture is concentrated under vacuum, dissolved in 20 mL CH₂Cl₂, then asolution of trityl amine (28.3 g 88.52 mmol) and Et₃N in 20 mL CH₂Cl₂ isadded dropwise. The reaction mixture is stirred for 48 h at roomtemperature.

The reaction is stopped by addition of saturated solution of K₂CO₃ andthe desired product extracted by Et₂O.

The organic layer is washed with K₂CO₃ sat., HCl 2M, dried over Na₂SO₄,and concentrated under reduced pressure. The crude product is eluted onsilica gel (elution CHex/AcOEt 6/4) yielded 20.9 g of the desiredcompound 12 as a white solid.

Yield: 65.0% Mp: 102–104° C. TLC (CHex/AcOEt 6/4) Rf:0.56 HPLC KromasilC18 5μ100A, 250×4.6 mm, CH₃CN—H₂O (0.05% TFA) 70-30 R_(t)=12.45 min ¹HNMR (CDCl₃ 270 MHz): 7.4–7.1(m, 15H), 6.6(s, 1H), 4.15 (q, 4H), 3.4 (t,1H), 2.35 (t, 2H), 2.1 (q, 2H), 1.3 (t,6H).

Step 4—Synthesis of Monoethyl 2-(2-N-tritylcarboxamidoethyl)malonate(13)

To a solution of compound 12 (20.93 g, 44.41 mmol) in 80 mL EtOH at 0°C. is added KOH (2.53 g 1.025 eq) in 100 mL EtOH. The reaction mixtureis stirred for 48 h at 4° C. The reaction mixture is concentrated underreduced pressure. The mixture is dissolved in water, extracted by Et₂O.The aqueous layer is acidified with HCl 3M. The precipitate is filtered,dried, given 16.4 g compound 13 as a white solid.

Yield: 65.0% Mp: 122–124° C. TLC (CHex/AcOEt 6/4) Rf:0.56 HPLC KromasilC18 5μ100A, 250×4.6 mm, CH₃CN—H₂O (0.05% TFA) 70-30 R_(t)=12.45 min ¹HNMR (CDCl₃ 270 MHz): 7.4–7.1(m, 15H), 6.6(s, 1H), 4.15 (q, 2H), 3.4 (t,1H), 2.35 (t, 2H), 2.1 (q, 2H), 1.3 (t,3H).

Step 5—Synthesis of Ethyl 2[2-(N-trityl)carboxamido ethyl)]acrylate (14)

Et₂NH (3.80 mL, 36.85 mmol), 37% sol. Formaldehyde (4.5 mL,1.5 eq) aremixed with compound 13 (16.4 g 36.85 mmol) and stirred 48 h at roomtemperature. The reaction mixture is taken up with 210 mL of a mixtureH₂O/Et₂O. The aqueous layer is extracted two times with Et₂O. Theorganic layers are washed with citric acid 10%, H₂O, NaHCO₃ sat., NaClsat., dried over Na₂SO₄, and concentrated under reduced pressure yieldedcompound 14 (11.71 g) as a white solid.

Yield: 79.6% Mp: 120–122° C. HPLC Kromasil C18 5μ100A, 250×4.6 mm,CH₃CN—H₂O (0.05% TFA) 7030 R_(t)=11.81 min ¹H NMR (CDCl₃ 270 MHz):7.4–7.1(m, 15H), 6.6(s, 1H), 6.1 (s, 1H), 5.6 (s, 1H), 4.14 (q, 2H), 2.6(t, 2H), 2.4 (t, 2H), 1.3 (t,3H).

SEQUENCE DATA SEQ ID No: 1-Peptide Sequence 1 1 AQENLQKIVD SLESSRAEREELYKWFHQHP EMSMQEHETS KRIAEELEKL GLEPQNIGVT 61 GQVAVIKNGE GPSVAFRADFDALPITENTG LDYSADPELG MMHACGHDLH TTALLGAVRA 121 LVENKDLWSG TFIAVHQPGEEGGGGARHMV DDGLAEKIAA PDVCFAQHVF NEDPAFGYVF 181 TPGRFLTAAS NWRIHIHGEGGHGSRPHLTK DPIVVAASII TKLQTIVSRE VDPNEVAVVT 241 VGSIEGGKST NSIPYTVTLGVNTRASNDEL SEYVQNAIKR IVIAECQAAG IEQEPEFEYL 301 DSVPAVINDE DLTEQLMAQFREFFGEDQAV EIPPLSGSED YPFIPNAWGV PSVMWGWSGF 361 AAGSDAPGNH TDKFAPELPDALERGTQAIL VAAAPWLMK SEQ ID No: 2-Peptide 2A-Q-E-N-L-Q-K-I-V-D-S-L-E-S-S-R-A-E-R-E-E-L-Y-K-W-F-H-Q-H-P-E-M-S-M-Q-ESEQ ID No: 3-Peptide 3 D-L-W-S-G-T-F-I-A-V-H-Q-P-G-E-E-I-G-G-T-K SEQ IDNo: 4-Peptide 4 W-G-W-S-G-F-A-A-G-S-D-A-P-G-N SEQ ID No: 5-NucleotideSequence 1 1 AATCGGGTCA TGGCACAGGA AAATTTGCAA AAGATTGTAG ATAGTCTCGAGTCCTCCCGC 61 GCGGAACGCG AAGAACTGTA CAAGTGGTTC CACCAGCACC CGGAAATGTCGATGCAGGAG 121 CACGAAACCT CCAAGCGCAT CGCAGAAGAG CTAGAGAAGC TCGGCCTTGAGCCGCAGAAC 181 ATCGGCGTGA CCGGGCAGGT CGCGGTAATC AAGAACGGTG AAGGCCCGAGCGTGGCATTT 241 CGTGCGGACT TTGATGCCTT GCCGATCACC GAGAACACCG GGCTGGATTACTCGGCGGAT 301 CCCGAGCTGG GCATGATGCA CGCCTGCGGC CACGATTTGC ACACCACTGCCCTACTCGGC 361 GCGGTGCGCG CGCTGGTGGA GAACAAGGAC CTGTGGTCCG GCACCTTCATCGCAGTCCAC 421 CAACCCGGTG AGGAAGGCGG CGGCGGGGCC CGCCACATGG TGGACGACGGCCTCGCGGAG 481 AAGATCGCGG CGCCGGATGT GTGTTTCGCC CAGCACGTGT TCAACGAAGACCCCGCCTTT 541 GGCTACGTGT TCACCCCCGG CCGGTTTCTA ACGGCGGCGT CGAACTGGAGAATCCACATC 601 CACGGCGAGG GCGGACACGG TTCCCGTCCG CACCTGACCA AGGACCCGATTGTGGTGGCG 661 GCCTCGATCA TTACCAAGCT GCAGACGATT GTCTCCCGCG AAGTCGATCCGAATGAGGTC 721 GCAGTGGTCA CCGTCGGCTC CATCGAGGGC GGCAAGTCCA CCAACTCGATCCCGTACACC 781 GTCACCCTCG GCGTGAACAC CCGAGCCTCC AACGATGAGC TCTCCGAGTACGTCCAGAAC 841 GCCATCAAGC GCATCGTCAT CGCGGAGTGC CAGGCTGCAG GCATCGAACAGGAGCCGGAA 901 TTCGAGTACC TGGACTCAGT CCCGGCCGTG ATCAACGACG AGGATCTCACCGAACAGCTC 961 ATGGCGCAGT TCCGGGAGTT CTTCGGCGAG GACCAGGCGG TAGAGATTCCGCCCCTGTCC 1021 GGCAGCGAGG ACTACCCCTT CATTCCGAAC GCCTGGGGCG TGCCGAGTGTGATGTGGGGA 1081 TGGTCCGGCT TCGCCGCAGG TTCTGACGCA CCGGGCAATC ACACCGACAAGTTCGCCCCC 1141 GAGCTTCCAG ATGCCCTCGA ACGCGGCACC CAGGCCATTC TGGTGGCCGCCGCGCCCTGG 1201 TTGATGAAGT GA SEQ ID No: 6-Nucleotide Sequence 2 1GGGCAGCCGG CTCACGTGGC GTGAGCGAGC GAGACCTTCG GTCGATTACC GCACCGAAAG 61GAACCCCTGT GAGCGAAGCT CTCCGCGAAG AACAGCGCCT GCTCGAGCGC TTCATGTGGC 121TTTCGACCAT TGCCTCCATC TTTGCCATTG CGCTGAAGCT GTACGCGGCG TGGGTGACGG 181GCTCGGTCGG CTTTTTCTCC GACGCGATCG AGTCCTTTGC CAACCTGGCC GCTGCGGTGG 241TGGGGCTTTG GGCGCTGAAG CTCTCGGCCA AACCGGCCGA TGCCAACCAC AATTTCGGCC 301ATGCCAAGGC GGAATACTTC GCGGCGCAGG TGGAAGGCAC GATGATTCTG GTGGCCTCCG 361TGGTCATCAT CGTCACCGCC GTGCAGCGCA TCATCGACCC GGCTCCGCTT AACCAGCTCG 421GGATCGGCCT GGTTTTCTCC GTTGTTGCCA CCGTGATCAA CCTCGGCGTC GGCGTCGCGC 481TGGTGCGGGC GGGTCGCACC CACCGCTCCA GCACACTCGA GGCCGATGGA AAGCATTTGC 541TTACCGACGT CTGaACCACC GTGGGAGTCA TCGCCGGCAT GGCGTTGGTG TGGCTGACGG 601GGTGGAACGT CTTGGACCCC ATCGTGGCGT TGATTGTCGG TGCCAACATC CTCTTCACGG 661GATACCACTG TTGCGCCAGG CGATGATGGG GCTGCTCTCC GAGGCGCTGC CGAGAGACGA 721GGTCGAGACC GTGCAGGGGT TCTTGGACGG GTTCGCGGCA GAGCACGGCG TGGCGTTCAC 781TTCGCTGCGC ACCTCGGCGT TTGGCCGCGA CCGCCTCATC AACGTCGTGA TGCAGGTTCC 841CGGCGAATGG TCTGTGGAGG CCTCGCACGA GTACGCGGAC CAGGTCGAGG TGGGCATCGC 901TACCGCGCTG GGGCACGCCG AAACCATCGT GCACATCGAA CCGCTTGGAC ATCACACCAA 961AACAGGCCCC ATGGCGGTGT AGTAACCGCC GTAGAATCGG GTC SEQ ID No: 7-NucleotideSequence 3 AAG UGG UUC CAC CAG CA SEQ ID No: 8-Nucleotide Sequence 4 TCYTCD CCN GGC TGG TG (Y = C/T; D = A/G/T; N = A/C/G/T) SEQ ID No:9-Nucleotide Sequence 5 TCR TTN GGR TCV ACY TC (R = A/G; V = A/C/G; Y =C/T; N = A/C/G/T) SEQ ID No: 10-Nucleotide Sequence 6 CTT CAC CGT TCTTGA TTA CCG GGA CCT SEQ ID No: 11-Nucleotide Sequence 7 CTC TAG CTC TTCTGC GAT GCG CTT GGA SEQ ID No: 12-Nucleotide Sequence 8 CCG CAC CTG ACCAAG GAC CCG ATT GTG SEQ ID No: 13-Nucleotide Sequence 9 CCT CGA TCA TTACCA AGC TGC AGA CGA SEQ ID No: 14-Nucleotide Sequence 10 CAT GCC ATG GCACAG GAA AAT TTG CAA SEQ ID No: 15-Nucleotide Sequence 11 CCC AAG CTT TCACTT CAT CAA CCA GGG CG

1. An isolated N_(α)-acyl-glutamine-aminoacylase comprising an amino acid sequence, which has a sequence identity of at least 95% to the amino acid sequence set forth in SEQ ID NO:
 1. 2. An isolated enzyme comprising the amino acid sequence set forth in SEQ ID NO:
 1. 3. An isolated enzyme encoded for by the nucleic acid comprising a the nucleotide sequence set forth in SEQ ID NO:
 5. 4. A method of screening compounds for inhibitor activity of an enzyme as defined in claim 1 comprising the steps of I) incubating the enzyme of claim 1 or cell extract containing said enzyme with its substrate and a compound with potential inhibitory properties, and II) measuring release of malodorous 3-hydroxy-3-methyl-hexanoic acid or 3-methyl-3-hexanoic acid compounds and/or free L-glutamine. 